Bv510 streptavidin

Manufactured by BioLegend

BV510 streptavidin is a fluorescent labeling reagent used in flow cytometry applications. Streptavidin is a tetrameric protein that binds to biotin with high affinity, allowing it to be used to detect and analyze biotinylated molecules. The BV510 fluorescent dye is conjugated to the streptavidin, providing a bright signal for detection.

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7 protocols using bv510 streptavidin

SARS-CoV-2 Spike Protein-Specific B Cell Detection

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To detect antigen-specific B cells, approximately 5 × 10⁵ PBMCs were stained with 100 ng full-length biotinylated spike protein (Sino Biological) preincubated with Streptavidin-BV510 (Biolegend) at a 2:1 ratio for 1 hour at 4°C to ensure maximum staining quality before surface staining with CD20-FITC (Biolegend, 2H7) for an additional 30 minutes. Streptavidin PE (Biolegend) was used as a decoy probe to gate out SARS-CoV-2 nonspecific streptavidin binding. Samples were washed twice and resuspended in 200 μL FACS buffer before being analyzed on Attune NxT (Life Technologies).

Sureshchandra S., Lewis S.A., Doratt B.M., Jankeel A., Coimbra Ibraim I, & Messaoudi I. (2021). Single-cell profiling of T and B cell repertoires following SARS-CoV-2 mRNA vaccine. JCI Insight, 6(24), e153201.

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Flow Cytometry Analysis of OT-II Cells and DCs

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The following antibodies and reagents were used: anti-CD4 (RM4-5; Biolegend/eBioscience), -CD11c (N418; BD/Biolegend), -CD44 (IM7; Biolegend), -CD45.1 (A20; Biolegend), -CD62L (MEL-14; Biolegend), CD127 (A7R34; BD/Biolegend), -KLRG-1 (2F1; Biolegend/eBioscience), -Ki67 (SolA15; eBioscience), -I-A^b (AF6-120.1; Biolegend), -Thy1.1 (OX-7; Biolegend), -TCR Vα2 (B20.1; Biolegend), streptavidin-BV510 (Biolegend) and streptavidin-PE (Biolegend). For intranuclear staining of Ki67, cells were first stained with the indicated antibodies directed against cell surface molecules. Afterwards cells were fixed with the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer's instructions and subsequently incubated with anti-Ki67 for 30 min at 4°C. Samples were measured on LSRFortessa flow cytometer (Becton Dickinson) and analyzed by FlowJo 9 and 10 software (FlowJo, LLC). To calculate the fold expansion of OT-II cells or DCs, the respective cell populations were quantified. For each experiment a mean value was calculated for the Rag^WT group. Finally, cell numbers of individual mice, including Rag^WT mice, were calculated in relation to the mean value of the Rag^WT group. Relative mean fluorescence intensities (MFIs) and relative frequencies of OT-II cells or DCs were calculated in analogy.

Knop L., Frommer C., Stoycheva D., Deiser K., Kalinke U., Blankenstein T., Kammertoens T., Dunay I.R, & Schüler T. (2019). Interferon-γ Receptor Signaling in Dendritic Cells Restrains Spontaneous Proliferation of CD4+ T Cells in Chronic Lymphopenic Mice. Frontiers in Immunology, 10, 140.

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Isolation of mouse hematopoietic progenitor cells

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Mouse bone marrow cells were collected from the femurs, tibiae and iliac crest and depleted of red blood cells by an ammonium chloride lysis step (STEMCELL Technologies). Cells were lineage depleted using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (19856, STEMCELL Technologies). Lineage⁻ c-Kit⁺ (LK) cells were then isolated using the following antibodies (clone and company): streptavidin BV510 (BioLegend), c-kit APC-Cy7 (2B8, BioLegend), Sca1 BV421 (D7, BioLegend), CD45 FITC (30-F11, BioLegend), EPCR (CD201) PE (RMEPCR1560, STEMCELL Tech), CD150 PE/Cy7 (TC15-12F12.2, BioLegend), CD45 FITC (30-F1,1 BD Bioscience) and CD48 APC (HM48-1, eBioscience). Flow cytometry was performed on an LSRII Fortessa (BD) and all data were analyzed using FlowJo (BD). A representative gating strategy is shown in Figure S12.

Isobe T., Kucinski I., Barile M., Wang X., Hannah R., Bastos H.P., Chabra S., Vijayabaskar M.S., Sturgess K.H., Williams M.J., Giotopoulos G., Marando L., Li J., Rak J., Gozdecka M., Prins D., Shepherd M.S., Watcham S., Green A.R., Kent D.G., Vassiliou G.S., Huntly B.J., Wilson N.K, & Göttgens B. (2023). Preleukemic single-cell landscapes reveal mutation-specific mechanisms and gene programs predictive of AML patient outcomes. Cell Genomics, 3(12), 100426.

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Immunostaining and Clearing of Pancreatic Tissue

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Thick 100 μm cryostat sections were rehydrated in PBS. Sections and whole mount pancreata were blocked overnight in PBS, 2% donkey serum and 0.5% Triton-100X. The samples were incubated in primary antibodies (Chromogranin A, 1:180 from Abcam, ab15160; insulin, 1:100, from Abcam, ab7842; glucagon 1:200, from Abcam, ab10988); and Dolichos biflorus agglutinin (DBA) biotinylated (1:270, from Vectorlabs, B-1035) for 3 days at 4 °C. Secondary antibodies were applied (from Thermo Fisher Scientific) and AF647-Streptavidin (1:800, from Thermo Fisher Scientific, S21374), BV510-Streptavidin (1:600, from Biolegend, 405233) for 2 days at 4°C. All tissues were cleared with RapiClear 1.52 (from SunJin Lab, RC152001).
Thin sections were incubated in 2% Serum, 0.1% Triton-100X in PBS for 30 min, subsequently incubated in primary antibody in 0.01% Triton-100X in PBS overnight (insulin 1:100, from Abcam, ab7842, glucagon 1:200 from Abcam, ab10988, cleaved caspase 3, 1:400, from Cell Signalling, 9664S), washed 3 times for 10 min the next day, and incubated with secondary antibody in 0.01% Triton-100X for 3 h, washed and mounted.

Sznurkowska M.K., Hannezo E., Azzarelli R., Chatzeli L., Ikeda T., Yoshida S., Philpott A, & Simons B.D. (2020). Tracing the cellular basis of islet specification in mouse pancreas. Nature Communications, 11, 5037.

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Assessing CD8+ T Cell Cytolytic Potential

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Rested ex vivo CD8⁺ T cells were assessed for CMV and
HIV specificity using HLA-matched tetramers. Tetramers were made via
biotinylated monomer conjugation to BV510 streptavidin (BioLegend). Monomers
were obtained from Dr. Soren Buus (University of Copenhagen). The A02-NV9
tetramer was used to stain for CMV-specificity while pools of A02-SL9, B27-KK10,
B53-QW9, B53-YY9, B57-IW9(p24), B57-IW9(RT), B57-KF11, B57-QW9, and B57-TW10
tetramers were used to stain for HIV-specificity. Subsequent surface stains
included anti-CD62L-PE-Cy5 (clone DREG-56; BioLegend), anti-CD3-AF700 (clone
HIT3a; BioLegend), anti-CD8-FITC (clone SK1; BioLegend), anti-CD45RA-BV605
(clone HI100; BioLegend), and LIVE/DEAD Blue. Following permeabilization, the
cells were intracellular stained using anti-perforin PE/Cy7 (clone B-D48;
BioLegend), anti-granzyme A-PerCp/Cy5.5 (clone CB9; BioLegend), anti-granzyme
B-Pacific Blue (clone GB11; BioLegend), anti-granzyme H (biotinylated and
secondary stained with BV650 streptavidin) (polyclonal antibody, R&D),
anti-granzyme K-PE (clone GM26E7; BioLegend), and anti-granzyme M-eFluor660
(clone 4B2G4; eBioscience). The cells were analyzed by flow cytometry.
Naïve CD8⁺ T cells within the mixed population of
CD8⁺ T cells were used as internal gating controls for
perforin and each granzyme as these cells do not express these effector
molecules.

Clayton K.L., Collins D.R., Lengieza J., Ghebremichael M., Dotiwala F., Lieberman J, & Walker B.D. (2018). Resistance of HIV-infected macrophages to CD8 T lymphocyte-mediated killing drives immune activation. Nature immunology, 19(5), 475-486.

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Quantifying SARS-CoV-2-specific Memory B Cells

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SARS-CoV-2-specific memory B cells were detected as we described previously^{11 (link)}. First, biotinylated antigens were multimerized with fluorescence-labeled streptavidin individually. Recombinant spike protein (R&D, #BT10549-050) was mixed with BV510-streptavidin (BioLegend) at 10:1 ratio and BV785-streptavidin (BioLegend) at 18:1 ratio at 4 °C for 1 h. Recombinant RBD protein (R&D, #BT10500-050) was mixed with BV421-streptavidin (BioLegend) at 20:1 ratio at 4 °C for 1 h. The antigen probes prepared above were then mixed in 50 mM free d-biotin (Macklin) in PBS to ensure minimal cross-reactivity. PBMCs were thawed and let to rest at 37 °C with 5% CO₂ for 2 h and stained with Zombie Red (BioLegend) in PBS at room temperature for 20 min. Cells were then stained with 50 μL of antigen probe cocktail containing 100 ng of spike and 50 ng of RBD at 4 °C for 30 min. Cells were washed with PBS and then stained with the following antibody cocktail: anti-CD3-Pacific Blue™, anti-CD19-PE-CY7, anti-CD27-AF700, anti-IgD-FITC, anti-CD38-BV650, anti-IgM-BV605, anti-IgG-AF647, anti-IgA-PE all from BioLegend at 1:100 dilution. Samples were acquired by flow cytometry.

Liu Y., Zeng Q., Deng C., Li M., Li L., Liu D., Liu M., Ruan X., Mei J., Mo R., Zhou Q., Liu M., Peng S., Wang J., Zhang H, & Xiao H. (2022). Robust induction of B cell and T cell responses by a third dose of inactivated SARS-CoV-2 vaccine. Cell Discovery, 8, 10.

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Assessing CD8+ T Cell Cytolytic Potential

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